Project summary Nuclear pore complexes (NPCs) are multi-protein assemblies embedded in the nuclear envelope, forming channels that mediate the regulated bidirectional nucleocytoplasmic transport of macromolecules. This transport allows for communication between the central organelle, the nucleus, and the rest of the cell, providing a crucial means to control gene expression, signaling networks, and cell homeostasis. Due to its central role, even modest disruption of the NPC has profound effects on cellular function, leading to many cancers such as leukemia and sarcomas. Crucially, a major causative connection in cancer and the NPC is the communication between the nucleus and the mitochondria in the apoptotic signaling pathway, triggered by intracellular damage or by oncogenic stress. The examples of Selinexor and Verdinexor have now established nucleocytoplasmic trafficking as a valid and powerful anti-cancer drug target; and there is strong evidence that at least in part these drugs are effective because they regulate this nuclear-mitochondrial communication pathway. We hypothesize that cancer- associated NPC alterations change the NPCs? structure and interactome that in turn impact their overall capability to serve in transport and intracellular communication. The proposed study is divided into two independent but synergistic aims that will decipher the structural and functional defects caused by these oncogenic alterations, focusing on downstream effects on the nuclear-mitochondrial apoptotic signaling pathway. The first Aim (1.1) is to characterize the changes in NPC interactome caused by oncogenic Nup alterations; specifically the overexpression of Nup62 or Nup88 or presence of TPR-FGFR1 or Nup214-Abl1 fusion mutations. We will establish a tissue culture model system expressing each of these cancer-associated Nup alterations and then compare their interactomes to the normal state using affinity-capture and mass spectrometry methodologies already established in our lab. As an additional tool, we suggest (Aim 1.2) to produce novel research tools in the form of nanobodies against cancer-associated Nups. Importantly, Aim 1.1 is not dependent on Aim 1.2. Aim 2 will focus on the functional impact of the oncogenic alterations. We will first (Aim 2.1) examine how these alterations affect nuclear and mitochondrial morphology and behavior. First we will assess changes in cellular morphology and behavior in cell lines bearing oncogenic Nup alterations. Next, we will assess how the Nup oncogenic alterations affect the mitochondria. Finally (Aim 2.2), we will look for changes in nucleocytoplasmic trafficking under oncogenic conditions and how it affects nuclear ? mitochondrial communication. We will monitor for changes in protein import and export, localization of transport factors and we will look for changes in specific mediators of nuclear-mitochondrial signaling. Our techniques and tools will (i) allow us to identify disease-causing alterations in NPC architecture, (ii) detect altered amounts or localizations of Nups due to these alterations, and correlate (i) with (ii) to determine the underlying mechanism of the Nup-induced oncogenic changes.